The goal of this project is to understand transmission and phosphorylation-induced modulation of the calcium signal in normal and diseased heart. The dynamics of heart contraction are controlled by calcium-mediated troponin/tropomyosin interactions that regulate force-generating interactions between actin and myosin. Cardiac troponin (cTn) is the calcium sensitive switch consisting of troponin C (cTnC), troponin I (cTnl), and troponin T (cTnT). Unique isoforms of the cardiac subunits allow modification of heart contraction during development and disease. In the heart, phosphorylation of the cTnl and cTnT components help modulate contractility. This proposal will fill critical gaps in our molecular understanding of cardiac regulation and modulation by phosphorylation of the cTnl component. The structural consequences of phosphorylation at the major sites on cTnl (Ser23/24, Ser43/45, and Thr144) will be investigated. Altered phosphorylation can lead to specific disease states such as familial hypertrophic and dilated cardiomyopathies and end stage heart failure. The general hypothesis is that understanding phosphorylation-induced conformational events in cardiac troponin will provide insights into the pathologies of specific disease states and aid in the design of pharmacological agents capable of modulating contraction through alternations in thin filament protein interactions. The experiments proposed are designed to fill critical gaps in our structural knowledge of cardiac thin filament regulation by charge-induced modification of cTnl at the major phosphorylation sites. NMR supplemented with small-angle x-ray and neutron scattering will permit us to characterize global and atomic level structural and dynamic information at cTn subunit interfaces. Our aims focus on investigating the molecular determinants and conformational states between cardiac troponin subunits responsible for modulating the calcium signal as a consequence of cTnl phosphorylation. Aim 1: Decipher the role of bisphosphorylation of the cardiac specific N-extension of cTnl in the calcium switch mechanism. Aim 2: Does phosphorylation of the N-domain of cTnl modulate Ca2+/Mg2+ -dependent protein-protein interactions and dynamics critical to modulating the hearts response to calcium? Aim 3: Identify and correlate Thr144 phosphorylation, located in the inhibitory region of cTnl, induced structural changes leading to altered myofilament response. PERFORMANCE SITE(S) (organization, city, state) University of Cincinnati College of Medicine Cincinnati, OH PHS 398 (Rev. 04/06) Page 2 Form Page 2